Sexually Dimorphic Responses Reveal Multifaceted Benefits of Glibenclamide in Traumatic Brain Injury

Anupama Rani; Sudhanshu P Raikwar; Wonsuk Yoo; Saif Ahmad; Vincent A Vagni; Keri L Janesko-Feldman; Shaun W Carlson; Adam Eberle; Margaux Miller; John Helm; Joshua Catapano; Benjamin E Zusman; Shashvat Desai; Raemier Anne M Javelosa; Semeon Afework; Erin Audrey McNally; Gary Kohanbash; Dhivyaa Rajasundaram; Michael F Waters; Andrew Ducruet; Ashutosh Jadhav; Aditya Kumar; Chia-Ling Phuah; Patrick M Kochanek; Ruchira M Jha

doi:10.1089/neu.2024.0232

. 2025 Jun 12;42(15-16):1319–1344. doi: 10.1089/neu.2024.0232

Sexually Dimorphic Responses Reveal Multifaceted Benefits of Glibenclamide in Traumatic Brain Injury

Anupama Rani ^1,^†, Sudhanshu P Raikwar ^1,^†, Wonsuk Yoo ^1,², Saif Ahmad ¹, Vincent A Vagni ^3,⁴, Keri L Janesko-Feldman ^3,⁴, Shaun W Carlson ^4–6, Adam Eberle ¹, Margaux Miller ¹, John Helm ¹, Joshua Catapano ⁷, Benjamin E Zusman ⁸, Shashvat Desai ⁷, Raemier Anne M Javelosa ¹, Semeon Afework ¹, Erin Audrey McNally ¹, Gary Kohanbash ^9,⁶, Dhivyaa Rajasundaram ^9,¹⁰, Michael F Waters ¹¹, Andrew Ducruet ⁷, Ashutosh Jadhav ⁷, Aditya Kumar ⁷, Chia-Ling Phuah ¹², Patrick M Kochanek ^3,^4,^9,¹³, Ruchira M Jha ^1,^7,^12,^*

PMCID: PMC12431513 PMID: 40510018

Abstract

Sex disparities in traumatic brain injury (TBI) remain poorly understood. Previous data suggest that males are more susceptible to acute secondary injury processes and cell death, whereas females are more vulnerable chronically. Additional sex-based differences have been reported depending on injury model/severity and post-traumatic neurodegeneration. This gap in understanding limits therapy translation. We previously demonstrated sex-based differences in genetic modulation of a key pathway of secondary injury in TBI, sulfonylurea-receptor 1 (SUR1). Glibenclamide (GLI, SUR1-inhibitor) has shown promise in pre-clinical and early clinical studies of TBI and stroke. Here, we evaluated GLI’s modulation of multifaceted TBI outcomes across sex for the first time. In total, 120 mice were randomized to controlled cortical impact (CCI) ± GLI or vehicle (dimethyl sulfoxide, DMSO). Either vehicle or GLI treatment was administered post-CCI using an intraperitoneal (IP) loading dose (10 µg/mouse, 10 min post-TBI), followed by a 7-day subcutaneous maintenance infusion at 0.5 µL/h using ALZET mini-osmotic pumps (1007D, Durect Corp.). Mice were tested for cognitive function (Morris water maze, MWM), motor function (rotarod), anxiety (elevated plus maze, EPM), immunofluorescence markers of neurodegeneration (TAU, TDP43), neurogenesis (SOX2, Ki67), angiogenesis (VEGFA), and cerebral blood flow (CBF) to interrogate behavioral, molecular, and physiological effects of TBI and therapy. Different measures within behavioral, immunofluorescence, and CBF outcomes varied across sex, either post-CCI and/or in response to GLI. Motor impairment had baseline differences across sex post-CCI. In both sexes, behavioral deficits were improved by GLI. The effect of GLI on behavior was moderated by sex, with greater benefit in males versus females, including improved MWM latency (p_{treatment*sex_interaction} < 0.0001) and rotarod latency (p_{treatment*sex_interaction} = 0.016, revolutions per minute, p_{treatment*sex_interaction} = 0.03). Males had increased anxiety post-CCI (EPM); GLI was beneficial across sexes. TDP43 and TAU in several brain regions were increased 72 h post-CCI (males>females, all p < 0.0001). These remained markedly elevated only in females by 21 days, whereas TAU in males decreased without treatment. GLI downregulated TDP43 and TAU across sex and brain region (all p < 0.01–0.0001). In females only, DMSO had similar effects as GLI on TDP43 and TAU. SOX2 was increased in the dentate gyrus (DG) only in males post-CCI (p_72h < 0.01, p_21d < 0.001). GLI increased DG SOX2 in females (p_72h < 0.05, p_21d < 0.001). GLI increased VEGFA at 72 h across sexes. CCI reduced CBF acutely in both sexes; in males, GLI improved this by 21 days (p = 0.031). In females, both GLI and DMSO-vehicle benefited CBF versus untreated-CCI. We demonstrate novel sex-based differences post-CCI and GLI-response across several metrics. TAU was chronically elevated (and responsive to treatment) in females, not males, potentially providing a sex-specific target. DMSO may have previously unrecognized benefits on certain pathways (TAU, CBF) in females. Although GLI has multifaceted benefits across sexes, effects are more pronounced in males. This may have important implications for clinical trial study design and analysis.

Keywords: cerebral blood flow, cognitive outcome, neurodegeneration, neurogenesis, sex differences

Introduction

Traumatic brain injury (TBI) is a growing health concern that leads to significant morbidity and mortality—it is projected to remain the leading cause of neurological disability in 2030.^1,2 Currently, there are no approved therapies that improve outcome post-TBI. In part, this is thought to be due to a mismatch between disease heterogeneity and treatment homogeneity impeding successful translation.¹ Sex-based biological differences are a basic but critical contributor to heterogeneity post-TBI with immediate clinical relevance.^3–5 These remain poorly understood and highly nuanced. There is a discrepancy between human versus animal studies: the former overall identify worse outcomes in females, whereas animal data frequently report the reverse, although data in females are limited in pre-clinical research.³ A more granular evaluation suggests that this is multifactorial, encompassing socioeconomic and biological factors as well as differences in outcome metrics used. Biologically, in pre-clinical models, although males are more susceptible to acute secondary injury processes and cell death, females are more vulnerable chronically.^6,7 Additionally, sex-based differences have been suggested in terms of the molecular pathways involved in the neuroinflammatory response (e.g., interferon-gamma, cyclooxygenase), TBI model/severity, and post-traumatic neurodegeneration, with suggestion that greater injury severity is associated with improved outcomes in females.³

We previously demonstrated marked sex-based differences in genetic modulation of sulfonylurea-receptor 1 (SUR1), transient receptor potential cation channel subfamily M member 4 (TRPM4), a key channel that is central to several forms of secondary injury in TBI.⁸ Additionally, SUR1 knockout has sex-based differences, with greater benefit in males in terms of impact on lesion volume, and the extent of benefit may depend on TBI endotype,^8,9 thus providing a potential opportunity for precision-medicine approaches to the problem of TBI heterogeneity. Importantly, this channel is clinically targetable—an existing sulfonylurea drug traditionally used to treat diabetes, glibenclamide (GLI), inhibits the SUR1-TRPM4 channel. GLI has shown exciting potential in pre-clinical and early clinical studies of TBI and stroke.^1,9–22 However, the utility of GLI is yet to be demonstrated in either a large phase-II or phase-III clinical trial. Many promising therapies in TBI have failed at this stage, in part possibly due to an incomplete preceding pre-clinical characterization of important/clinically relevant heterogeneity, including sex. Here, we evaluate the effect of GLI treatment on multifaceted TBI outcomes across sex for the first time.

The clinical focus of GLI has been as a potential treatment for acute post-TBI pathology like intracranial hypertension, cerebral edema, and contusion expansion. There have also been increasing data on its efficacy in other forms of acute neurological injury like ischemic stroke, intracerebral hemorrhage, subarachnoid hemorrhage, and spinal cord injury across species, including humans, although not all studies support benefit.^17,23–42 Aside from the acute impact, there is some suggestion of potential long-term neuroprotective effects with GLI, including cognitive and motor benefit, however, those outcomes remain to be fully investigated.^11,20,21 This is particularly important because of increasing evidence suggesting that a single moderate to severe TBI or repetitive mild TBI may lead to Alzheimer’s disease (AD)-like neuropathology and neurocognitive decline.^43–45 Neurodegeneration (specifically TAU deposition) has also been associated with cerebrovascular and blood flow dysfunction after TBI.⁴⁶ Accelerated neurocognitive decline may result from increased neurodegeneration, or impaired neurogenesis, particularly in the hippocampal DG. TAU deposition is also associated with CBF disturbances post-TBI,⁴⁶ and accumulation of TAU and aggregation of TAR DNA-binding protein 43 (TDP43) are important neuropathological features in TBI patients^47–51 linked to progressive memory decline.^45,52,53

Given the clinical interest of evaluating GLI in TBI trials, combined with the preponderance of pre-clinical and clinical research suggesting benefit, it is imperative to test and understand the response to this drug in both sexes. Sex-based regional differences in protein expression and physiology post-TBI could either increase or moderate the clinical response to GLI and inform future druggable targets. They have not previously been explored. We hypothesized that sex heterogeneously impacts multiple outcome measures after TBI in mice, and that it would influence the potential benefits of GLI treatment. In addition to functional studies, we evaluated effects on molecular and physiological outcome measures, including regional and temporal expression of markers (neurodegeneration, neurogenesis, angiogenesis) and CBF, since these may provide insight into the ultimate impact on functional recovery.

Materials and Methods

Animals

Adult male and female C57BL/6J mice (aged 12–14 weeks, Jackson Laboratory, Bar Harbor, ME) were used in all the experiments. All experiments and procedures were approved by the Institutional Animal Care and Use Committee at the Barrow Neurological Institute (Phoenix)/St. Joseph’s Hospital and Medical Center (Phoenix), and mice were housed in the Barrow Neurological Institute vivarium, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Mice were housed in a temperature-controlled animal facility on a 12-h light/dark cycle, and food and water were provided to the mice ad libitum. Mice were acclimated to the housing facility for at least 1 week before use. Experiments were randomized, and outcomes were obtained by investigators blinded to both treatment group and sex.

Experimental groups

A total of 120 mice (male n = 60; female n = 60) were used. For functional outcome studies, 48 male and female mice were block randomized into four groups (n = 6/group): naive, CCI, CCI + vehicle, and CCI + GLI. For CBF studies, 72 male and female mice were randomized as described earlier, into the same four groups (n = 3/group) for testing at 24 h, 72 h, and 21-day post-CCI. Mice in all the groups were euthanized at 24 h, 72 h, and 21 days via transcardiac perfusion with ice-cold phosphate-buffered saline (PBS, 0.1 M, pH 7.4) followed by 4% paraformaldehyde in PBS,⁵⁴ and brain sections were processed for immunofluorescence studies.

Controlled cortical impact

Controlled cortical impact (CCI), a standard clinically relevant model of TBI, was performed as previously described.^8,9 The injury device consists of a microprocessor-controlled pneumatic impactor, regulated by compressed air, with a 3-mm-diameter tip (TBI-0310 impactor, Precision System and Instrumentation, LLC). Anesthesia was induced and maintained with 4% and 2% isoflurane, respectively, using SomnoSuite low-flow anesthesia system for mice. The mouse was placed on a heated pad, and core body temperature was maintained at 37°C. The head was mounted in a stereotaxic frame (Kopf Instruments), and the surgical site was clipped and cleaned with povidone iodine and ethanol scrubs. A 10-mm midline incision was made over the skull, and a micromotor drill (Stoelting) was used to make a 5-mm craniotomy over the left parietal cortex. The bone flap was removed. TBI was induced by delivering CCI with a 3-mm diameter pneumatic impactor (velocity = 5.0 m/s, depth = 1.2 mm, dwell time = 50 msec). After injury, the bone flap was replaced and fixed immediately with dental cement to avoid any decompressive effects created by craniotomy, and the surgical site was closed by the silk sutures. Anesthesia was terminated, and the mouse was placed on a heated pad to maintain normal core temperature for 30–40 min postinjury. All animals were monitored after surgery every 15 min for 4 h and then daily.

Drug/vehicle administration

A stock solution of 2.5 μg/μL GLI (Sigma-Aldrich, Catalog no: G2539) was prepared by dissolving it in a solution of dimethyl sulfoxide (DMSO)/ethanol/H₂O:: 50/15/35 by volume. Vehicle contained only DMSO/ethanol/H₂O:: 50/15/35. As per our prior studies, either vehicle or GLI treatment was administered post-CCI using an intraperitoneal (IP) loading dose (10 µg/mouse, 10 min post-CCI), followed by a 7-day subcutaneous maintenance infusion at 0.5 µL/h using ALZET mini-osmotic pumps (1007D, Durect Corp.)⁹.

Morris water maze testing

Spatial learning and memory were assessed using the Morris water maze (MWM) test as previously described^55–57 to interrogate hippocampal function.^55,58,59 MWM training (postinjury days 15–19) was followed by a probe trial on 21 days to assess memory retrieval. A blue circular tank (diameter: 100 cm; walls: 60 cm high) was filled with water up to one-third height, maintained at a temperature of 24 ± 2°C. A plexiglass escape platform (9.5 cm × 35 cm) was submerged at a fixed position 1 cm below the water surface. Nontoxic white tempera paint was used to make water opaque. A ceiling camera directly over the water maze was connected to a computer with Ethovision XT 16 tracking software (Noldus, Wageningen, Netherlands). The maze was virtually divided into four arbitrary, equally spaced quadrants and distinct geometric visual cues were fixed in each quadrant at specific locations that were visible to mice during training and test. The training consisted of three trials each of 90 sec/day with an intertrial interval of 5 min for 5 days. During each trial, mouse was gently placed into the water, facing the wall of the pool, and was allowed to swim freely for 90 sec and locate the hidden platform. Mice failing to locate the platform within 90 sec were guided to the platform and allowed to remain on the platform for 15 sec. To prevent hypothermia, mice were gently dried with towel between and after the trials. Alterations in spatial learning and memory were assessed with standard metrics, including latency to platform (sec) and probe trials. Latency was defined as the time taken by the mouse to locate the hidden platform. In the probe trial, the hidden platform was removed, and the number of platform crossings was recorded.

Rotarod testing

The rotarod was used to assess motor coordination, grip strength, and balance.⁶⁰ A rotarod with touchscreen (76-0770, Panlab from Harvard Apparatus) was set up in a room with minimal disturbance. One day prior to the test (14 days postinjury), mice were acclimated to the moving rod for 120 sec, and three trials were conducted with intertrial interval of 10 min for each mouse. The rotarod was cleaned with 70% ethanol after each trial. On the day of test (15-day postinjury), mice were tested for latency to fall and revolutions per minute (RPM) at an accelerating rotation speed mode (4–40 RPM).^61,62 For each mouse, three trials were conducted for a maximum of 120 sec or until it fell from the rod. Briefly, each mouse was placed on the stationary rod for approximately 10 sec, then rod rotation was accelerated from 4 to 40 RPM over 120 sec, or until the mouse fell off the rotarod. Time from the start of acceleration until the mouse fell from rotarod and RPM resistance was automatically recorded by the software. Scores from the three trials on the day of test were averaged to yield a single score per mouse.

Elevated plus maze testing

An elevated plus maze (EPM) was used as described previously⁶³ to evaluate anxiety/risk-taking behavior using Ethovision XT 16 tracking software (Noldus, Wageningen, Netherlands). The apparatus consists of two enclosed (safe) arms and two open (aversive) arms, each 50 cm in length × 6 cm in width (Harvard Apparatus, USA), opposite from each other to create a plus shape with an open center (decision zone). The EPM was raised 85 cm above the floor. The test was performed at 21-day postinjury and consisted of a single trial. Mice were placed on the center platform, facing one of the closed arms, and allowed to freely explore the maze for 5 min. The maze was cleaned with 70% ethanol and wiped with paper towels between each animal. Behavioral metrics monitored were number and duration of entries into the open arm to quantify anxiety-like behavior (with fewer entries/less time in the open arm reflective of greater anxiety).^63,64

Cerebral blood flow assessment

Cerebral blood flow (CBF) was assessed using a blood perfusion imager (PeriCam PSI System, Perimed AB, Stockholm, Sweden) based on laser speckle contrast imaging (LSCI) technology as described previously.^65,66 Briefly, mice were anesthetized using isoflurane (4% induction, 2% maintenance), and body temperature was maintained at 37.0 ± 0.5°C. The skin of the anesthetized mice was cut to expose the skull at midline. Saline was applied to the exposed skull to normalize the effect of light refraction between mice, and the through-skull CBF was detected with speckle contrast. The dynamic and spatial distribution of CBF was then acquired and recorded from a 1 × 1 cm field of view using 785 nm, 80 mW laser with a sampling rate of 60 Hz at a working distance of 10 cm. A standardized region of interest (ROI) was used for each image in the ipsilateral and contralateral hemispheres to quantify perfusion units (PU). The CBF measurement was recorded for approximately 90 sec for each mouse (n = 3/group), naive, CCI, CCI + vehicle, and CCI + GLI at 24 h, 72 h, and 21-day postinjury. PUs were averaged over a 90 sec recording period for each mouse.

Brain processing for immunofluorescence assessments

Euthanasia was performed via transcardial perfusion with ice-cold phosphate-buffered saline (PBS, 0.1 M, pH 7.4) followed by 4% paraformaldehyde in PBS. Brains were postfixed in 4% paraformaldehyde overnight and then dehydrated in 30% sucrose in PBS for 24–72 h or till they sank to the bottom. Coronal cerebral cryosections were cut to 8 μm thickness and were stored at −20°C until processing.

Immunofluorescence

Standard immunofluorescence techniques were followed. Slides containing tissue sections were brought to room temperature and washed twice with PBS for 10 min. A hydrophobic barrier was made around the sections, which were washed twice again for 10 min with 1% normal donkey serum (NDS) in PBS with 0.4% Triton X-100 (PBST). To block nonspecific binding, sections were incubated with 5% NDS in PBST for 1 h at room temperature in a humidified chamber. Sections were incubated in primary antibodies diluted in 1% NDS in PBST at 4°C overnight, after which sections were rinsed three times in PBST for 10 min and incubated in a 1:800 dilution of the appropriate Alexa-Fluor secondary antibodies (Thermofisher) in 1% NDS in PBST for 1.5 h at room temperature. Sections were rinsed three times in PBST and mounted with Vectashield antifade mounting medium with DAPI (4',6-diamidino-2-phenylindole; Vector Laboratories). Images were captured at 20X magnification using a Keyence BZ-X800 Series All-in-One Fluorescence microscope, equipped with BZ-Image Analyzer software. Immunofluorescence intensity was quantified using BZ-Image Analyzer and was represented as integration value (IV). All analyses were performed by analysts blinded to treatment group.

Primary antibodies used include rabbit monoclonal NeuN (ab177487, Abcam; 1:200; RRID:AB_2532109), mouse monoclonal DCX (ab135349, Abcam; 1:200; RRID: AB_3096993), rat monoclonal Ki67 (14-5698-82, eBiosciences; 1:200; RRID:AB_10854564), mouse monoclonal SOX2 (MAB2018, RnD systems; 1:200; RRID:AB_358009), rabbit monoclonal TDP43 (ab109535, Abcam; 1:200; RRID:AB_10859634), mouse monoclonal TAU (MN1000, Thermofisher; 1:500; RRID:AB_2314654), rabbit monoclonal VEGFA (ab52917, Abcam; 1:200; RRID:AB_883427), rat monoclonal LYVE1 (MAB2125-100, RnD systems; 1:200; RRID: AB_3096995), and rabbit monoclonal angiopoietin-2 (ANG-2; MA5-32759, RnD systems; 1:200; RRID:AB_2810036). Secondary antibodies used include donkey anti-mouse IgG Alexa-Flour 568 (A10037; RRID:AB_11180865), donkey anti-rabbit IgG Alexa-Flour 488 (A21206; RRID:AB_2535792), and donkey anti-rat IgG Alexa-Flour 647 (A78947; RRID:AB_2910635) from Thermofisher Scientific.

Statistical analysis

The normality was examined graphically through the Q-Q plots for the continuous data, which were presented as mean ± standard errors of the mean. Generalized linear mixed model analysis and/or generalized estimation equations were used to evaluate longitudinal measures and the effect of treatment on functional outcome across sex and time. Behavioral, physiological, and molecular measures recorded at multiple time points (MWM training, rotarod, EPM, CBF, and immunofluorescence) were analyzed in linear mixed models with treatment and sex as fixed factors and time as a repeated measure. If the group comparisons were statistically significant, Tukey’s post hoc tests were performed for multiple comparisons procedure. Multiple group comparisons for different time points, sex, and effect of treatment for immunofluorescence data were performed by two-way ANOVA (Analysis of Variance) followed by Tukey’s post hoc correction test for multiple comparisons. A two-tailed test was considered statistically significant with p value of 0.05 as a cut point. R software (Version 4.2.1)⁶⁷ and Prism (V.9, GraphPad, San Diego, USA) were used for analyzing the data and to produce figures. Data are represented as mean difference (M.D.), 95% CI (Confidence Interval), and p value.

Results

Sexually dimorphic functional deficits post-CCI improved by GLI treatment

GLI-treated males had greater benefit than females on MWM post-CCI

Naive males and females had different MWM training patterns, where females had worse performance on 1 day and a steeper learning curve over 5 days (Fig. 1A, B). There were no sex differences in untreated mice post-CCI on MWM latency or probe trial (Fig. 1A–C, Tables 1 and 2). The response to GLI post-CCI, however, was different by sex, with evidence of greater benefit in males (Fig. 1A, B); this interaction of sex with treatment was statistically significant (p < 0.0001; Table 1).

FIG. 1. — Effect of GLI treatment on cognitive outcome, motor function, and anxiety across sex post-CCI. Cognitive outcome during training trials for 5 days in MWM and performance during probe trial in males and females with GLI treatment post-CCI are shown in **(A)** through **(C)**. **(Ai)** and **(Bi)** show performance of males and females mice from all the groups during training trials. **(Ci)** represents latency to reach the hidden platform on final day of test; (**Cii**) represents the number of platform crossings by males and females during probe trial test. (**D-i**, ii) Rotarod test data are represented as latency to fall and RPM. EPM test as depicted by decrease in the number of entries in open arm and duration in open arm (**E-i**, ii) in males and females. Linear mixed model results, including generalized linear mixed models (GLMM), are noted at the top of each subplot with the associated p values. Data are represented as mean ± SEM; data were compared using two-way ANOVA followed by Tukey’s post hoc test. During training **(A)** for males: At *2D = 0.02, 3D **p = 0.001, ***4D p = 0.0001, 5D ***p = 0.0001, CCI versus CCI + GLI, **(B)** for females: At 3D **p = 0.001, 4D p = 0.15, 5D *p = 0.03, CCI versus CCI + GLI for latency to platform. For **(C)**, **(D)**, **(E)**, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 Naive versus CCI, *p < 0.05, **p < 0.001, ***p < 0.0001 CCI versus CCI + GLI (M/F). CCI, controlled cortical impact; GLI, glibenclamide; RPM, revolutions per minute; 2D, day2; 3D, day3; 4D, day4; 5D, day5; M, male; F, female; SEM, standard errors of the mean.

Table 1.

Effect of GLI on Males and Females Post-TBI in MWM (5 Days Training)

Time points	Mean (±SE) males				GLMM p	Mean (±SE) females				GLMM p
Time points	Naive	TBI	TBI + Vehi	TBI + GLI	GLMM p	Naive	TBI	TBI + Vehi	TBI + GLI	GLMM p
D1	35.5 (4.9)	84.15 (4.8)	85.88 (2.7)	88.28 (1.1)	Treatment <0.0001* Time <0.001* Treatment × Time = 0.0094 Treatment × Sex <0.0001*	68.62 (8.3)	87.88 (2.1)	76.95 (4.3)	85.28 (3.1)	Treatment = 0.0076 Time <0.001* Treatment × Time = 0.0337* Treatment × Sex <0.0001***
D2	34.67 (7.1)	84.22 (4)	71.38 (6.8)	66.67 (7.3)		49.22 (11.6)	77.43 (7.9)	79.17 (6.8)	71.33 (3.8)
D3	33.05 (12)	81.00 (8.8)	50.57 (6.5)	46.17 (7.5)		48.82 (13.3)	83.00 (4.4)	75.10 (12.1)	53.12 (8.1)
D4	16.97 (2.6)	72.22 (11.2)	42.17 (7.2)	35.88 (11.5)		39.22 (8.6)	76.95 (9.4)	66.93 (11.8)	62.67 (10.5)
D5	13.05 (3.1)	70.9 (12.4)	49.23 (11)	29.35 (6.6)		25.95 (13.2)	74.88 (4.8)	53.55 (12.6)	51.20 (11.1)

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*p < 0.05.

**p < 0.01.

***p < 0.001.

GLI, glibenclamide; GLMM, generalized linear mixed model analysis; MWM, Morris water maze; TBI, traumatic brain injury.

Table 2.

Effect of GLI on Males and Females Post-TBI in MWM Test

Groups	Latency to platform (sec) mean (±SE) males	GLMM p	No. of platform crossings Mean (±SE) Males	GLMM p	Latency to platform (sec) mean (±SE) females	GLMM p	No. of platform crossings Mean (±SE) Females	GLMM p
Naive	10.5 (3.3)	Treatment <0.0007^*** Treatment × Sex = 0.27	9.50 (0.6)	Treatment = 0.11 Treatment × Sex = 0.17	20.67 (7.2)	Treatment = 0.0004^*** Treatment × Sex = 0.27	7.17 (1.6)	Treatment = 0.027^* Treatment × Sex = 0.17
TBI	79.33 (9.8)		1.50 (0.9)		62.67 (13.9)		0.33 (0.7)
TBI + Vehi	80.0 (9.9)		3.33 (1.1)		80.67 (9.3)		3.50 (1.8)
TBI + GLI	50.17 (15.2)		4.83 (1.6)		23.67 (5.5)		3.33 (1.1)

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*p < 0.05.

***p < 0.001.

GLI, glibenclamide; GLMM, generalized linear mixed model analysis; MWM, Morris water maze; TBI, traumatic brain injury.

CCI induced marked deficits in latency to platform versus naive in males that were corrected by GLI over 5 days (Table 1, Fig. 1A). Treatment effects were visible by 2 days. In females (Fig. 1B), there was no significant difference between CCI + GLI versus untreated-CCI at baseline on 1 days, however, by 2 days this started to diverge and improving with CCI + GLI versus untreated-CCI and became significant by 3 days (M.D. = 29.8; 95% CI = 12.3–47.4; p = 0.001). Over the course of 5 days, GLI-treated females performed better than untreated-CCI (M.D. = 23.67; 95% CI = 1.2–46.0; p = 0.03). However, although the benefit for GLI in females persisted at 5 days, the DMSO-vehicle had a similar benefit. This differed from males, where GLI outperformed DMSO-vehicle, and the latency to platform achieved by 5 days in GLI-treated males was shorter than in females (Fig. 1A).

The probe trial was performed 24 h after the 5 days of training, on 21 days post-CCI (Fig. 1Ci, ii). Here, untreated-CCI induced a significant deficit in latency to platform versus naive in both sexes (male: M.D. = −68.8; 95% CI = −113.8 to −23.87; p_male <0.0001, female: M.D. = −42; 95% CI = −80.8 to −3.17; p_female <0.01). There was no interaction of treatment with sex: treatment effects showed marked benefit (p_male= 0.0007, p_female=0.0004) in both sexes, driven by GLI (vehicle did not improve latency to platform vs. CCI in either sex; Fig. 1Ci). Treatment effects on the probe trial had a trend toward benefit in males but were significant in females with increased platform crossings versus untreated-CCI (M.D. = 39; 95% CI = 1.18–76.81; p < 0.05) (Fig. 1Cii). Mirroring the findings observed for the 5 days of MWM latency to platform testing, although DMSO-vehicle administration did not show benefit on probe trial performance in males, it had a benefit similar to GLI in females.

GLI improves rotarod deficits post-CCI in females and augments male performance beyond naive levels in males

Sex moderated the rotarod performance after both injury and treatment with GLI, including both latency to fall (p_{treatment_sex_interaction} = 0.03) and RPM (p_{treatment_sex_interaction} = 0.016; Fig. 1D, Table 3. Unlike the MWM findings, here, more profound motor deficits were noted only in females after CCI. In males, there was no difference in latency to fall and RPM in naive versus untreated-CCI, whereas in females both measures were significantly decreased (worsened) versus naive by CCI (latency: M.D. = 24.5; 95% CI = 6.4–42.5; p_latency = 0.001; RPM: M.D. = 5.6; 95% CI = 2.5–8.7; p_RPM <0.0001) (Fig. 1Di, ii).

Table 3.

Effect of GLI Treatment on Males and Females post-TBI in Rotarod (Motor Function)

Groups	Latency to fall (sec) mean (±SE) males	GLMM p	RPM mean (±SE) males	GLMM p	Latency to fall (sec) mean (±SE) females	GLMM p	RPM mean (±SE) females	GLMM p
Naive	42.87 (3.2)	Treatment <0.0001^*** Treatment X Sex = 0.030^*	10.62 (1.0)	Treatment = 0.0021^*** Treatment X Sex = 0.016^*	51.07 (6.4)	Treatment = 0.027^* Treatment X Sex = 0.030^*	13.7 (1.5)	Treatment = 0.03^* Treatment X Sex = 0.016^*
TBI	37.95 (1.4)		10.77 (0.3)		26.57 (1.0)		8.07 (0.2)
TBI + Vehi	54.17 (6.4)		10.28 (0.8)		58.57 (7.3)		10.82 (0.9)
TBI + GLI	88.43 (4.0)		14.50 (0.5)		72.18 (1.9)		12.28 (0.3)

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*p < 0.05.

***p < 0.001.

GLI, glibenclamide; GLMM, generalized linear mixed model analysis; TBI, traumatic brain injury.

Notwithstanding these baseline differences, both latency to fall and RPM increased with GLI in both sexes (Table 3, Fig. 1Di, ii). In females, latency to fall was increased (improved) not only versus untreated-CCI (CCI + GLI vs. CCI: M.D. = −45.6; 95% CI = −63.6 to −27.6; p < 0.0001) but also versus naive (CCI + GLI vs. naive: M.D. = −21.1; 95% CI = −39.1 to −3.1; p = 0.01). GLI improved RPM in females back to naive levels (CCI+GLI vs. CCI: M.D. = −4.2; 95% CI = −7.3 to −1; p = 0.001) (Fig. 1Dii). Although no deficits were noted in rotarod performance post-CCI versus naive in males, there was a treatment effect for both latency to fall (M.D. = −50.6; 95% CI = −68.6 to −32.5; p < 0.0001) and RPM (M.D. = −3.7; 95% CI = −6.8 to −0.59; p = 0.008). It suggests heightened motor resilience and enhanced performance with treatment, indeed GLI-treated males outperformed naive males.

GLI reduced anxiety measured by EPM across sex post-CCI

Sex did not significantly moderate the response to injury/treatment group on the number of entries (p_{treatment_sex_interaction} = 0.194) or duration (p_{treatment_sex_interaction} = 0.188) into the open arm on EPM (Fig. 1Ei, ii, Table 4). In males, CCI increased anxiety and produced a reduction in number (CCI vs. naive: M.D. = 5.3; 95% CI = 2.4–8.17; p = 0.0002) and duration (CCI vs. naive: M.D. = 26.6; 95% CI = 4.2–48.9; p = 0.01) of entries into the open arm. GLI treatment improved anxiety level as there was increase in number of entries (M.D. = −3.3; 95% CI = −6.1 to −0.4; p_{number_entries}=0.01). Although other parameter of EPM, that is, duration in open arm, showed an increasing trend with GLI treatment, but it was not significant. In females, although there was no benefit of either vehicle or GLI-treated mice versus untreated mice post-CCI in terms of increasing the number of entries into the open arm, the duration of time spent in the open arm was increased exclusively by GLI (CCI + GLI vs. CCI: M.D. = −33.8; 95% CI = −63.1 to −4.3; p = 0.02).

Table 4.

Effect of GLI Treatment on Males and Females Post-TBI in EPM (Anxiety Level)

Groups	No. of entries in open arm mean (±SE) males	GLMM p	Duration in open arm (sec) mean (±SE) males	GLMM p	No. of entries in open arm mean (±SE) females	GLMM p	Duration in open arm (sec) mean (±SE) females	GLMM p
Naive	5.83 (0.6)	Treatment = 0.0004^*** Treatment X Sex = 0.19	26.98 (7.3)	Treatment = 0.02^* Treatment X Sex = 0.18	7.83 (1.9)	Treatment = 0.27 Treatment X Sex = 0.19	22.27 (3.5)	Treatment = 0.03^* Treatment X Sex = 0.18
TBI	0.50 (0.4)		0.37 (0.3)		4.33 (1.3)		7.03 (2.3)
TBI + Vehi	2.83 (0.4)		13.55 (4.0)		4.33 (1.1)		24.68 (8.3)
TBI + GLI	3.83 (1.0)		16.33 (7.5)		5.0 (1.0)		40.85 (11.5)

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*p < 0.05.

***p < 0.001.

EPM, elevated plus maze; GLI, glibenclamide; GLMM, generalized linear mixed model analysis; MWM, Morris water maze; TBI, traumatic brain injury.

Sex- and region-specific differences in TDP43 and TAU expression post-CCI with or without GLI treatment

Given the sexually dimorphic impact of both CCI and GLI on functional outcome, we evaluated TDP43^45,47,49 and TAU expression in all groups and both sexes at 72 h and 21-day post-CCI across several brain regions—ipsilateral cortex, hippocampus (DG), and thalamus (Figs. 2, 4, Supplementary Fig. S1).

FIG. 2. — Heatmap visualization of neurodegeneration, neurogenesis, and angiogenesis markers with GLI treatment post-CCI Heatmap representation of expression of TAU, TDP43, SOX2, Ki67, and DCX in ipsilateral cortex in males **(i)** and females **(ii)** and effect of GLI treatment at 72 h and 21-day post-CCI. Heatmap representation of expression of TAU, TDP43, SOX2, Ki67, and DCX in ipsilateral DG in males **(iii)** and females **(iv)** and effect of GLI treatment at 72 h and 21-day post-CCI. Heatmap representation of expression of TAU, TDP43, in the thalamus in males **(v)** and females **(vi)** and effect of GLI treatment at 72 h and 21-day post-CCI. Heatmap representation of expression of SOX2, Ki67, and DCX in third ventricle in males **(vii)** and females **(vii)** and effect of GLI treatment at 72 h and 21-day post-CCI. The color legend is on the right of each heatmap. Purple indicates lower and yellow indicates greater protein expression levels. CCI, controlled cortical impact; GLI, glibenclamide.

Decreased 21 days DG, cortical, and thalamic TDP43 in GLI-treated mice across sex

TDP43 is involved in neuronal messenger ribonuclear protein (mRNP) granule metabolism with a recognized proteinopathy that contributes to neurodegeneration.^45,47,49 Post-CCI, TDP43 expression varied based on time-since-injury, sex, brain region, and treatment with GLI (Figs. 2i–vi and 3Ai, ii, Bi, ii, Ci, ii, Di, ii, Supplementary Figs. S2, S3, and S4).

TDP43 expression in the naive cortex, DG, and thalamus was higher in females versus undetectable in males (Fig. 3Bi, ii, Ci, ii, Di, ii, Supplementary Figs. S2, S3, and S4). TDP43 levels at 72 h postuntreated-CCI were elevated versus naive across sex in most brain regions (all p < 0.01); they were highest in the ipsilateral/pericontusional cortex in males and females.

In males, there was an increase in TDP43 expression in untreated-CCI versus naive, in the ipsilateral cortex at 72 h post-CCI (M.D. = −4.5E5; 95% CI = −5.8E6 to −3.3E6; p < 0.0001). Post-CCI, with GLI treatment, its expression decreased (CCI vs. CCI + GLI: M.D. = 1.9E6; 95% CI = 6.9E5–3.1E6; p = 0.0045) (Fig. 3Ai, Bi, Supplementary Fig. S2Ai). Similar pattern of TDP43 expression was observed at 21-day post-CCI with progressive decrease in TDP43 expression with GLI (CCI vs. CCI + GLI: M.D. = 1.0E6; 95% CI = 3.7E5–1.7E6; p = 0.005). In females, TDP43 expression in ipsilateral cortex was increased at 72 h post-CCI (untreated-CCI vs. naive: M.D. = −3.1E6; 95% CI = −3.8E6 to −2.4E6; p < 0.0001) (Fig. 3Aii, Bii, Supplementary Fig. S2Bi) but GLI had no effect. At 21-day post-CCI, GLI downregulated the increased TDP43 expression toward normal (CCI + GLI vs. untreated-CCI: M.D. =1.7E6; 95% CI = 8.0E5–2.6E6; p = 0.001; Table 5).

Table 5.

Effect of GLI Treatment on Protein Expression Post-TBI Across Sex and Time in Different Brain Regions

Brain regions	Cortex								Thalamus
Sex	Males				Females				Males				Females
Treatment	TBI^*		GLI^**		TBI^*		GLI^**		TBI^*		GLI^**		TBI^*		GLI^**
Time	72 h	21 days	72 h	21 days	72 h	21 days	72 h	21 days	72 h	21 days	72 h	21 days	72 h	21 days	72 h	21 days
Cell marker categories
Neurodegeneration
TAU	↑ p < 0.0001	↑ p < 0.0001	↓ p = 0.0032	↓ p < 0.0001	↑ p < 0.0001	↑ p = 0.0003	↓ p = 0.012	↓ p = 0.0006	No change	↑ p < 0.0001	No change	↓ p < 0.0001	↑ p < 0.0001	↑ p < 0.0001	↓ p < 0.0001	↓ p < 0.0001
TDP43	↑ p < 0.0001	↑ p = 0.0017	↓ p = 0.0045	↓ p = 0.005	↑ p < 0.0001	↑ p = 0.002	No change	↓ p = 0.001	↑ p < 0.0001	↑ p < 0.0001	↓ p < 0.0001	↓ p < 0.0001	No change	↑ p < 0.0001	↓ p = 0.002	↓ p < 0.0001
Angiogenesis
VEGFA	↑ p = 0.009	↑ p < 0.0001	↑ p < 0.0001	↓ p < 0.0001	↑ p = 0.007	↑ p < 0.0001	↑ p = 0.003	↓ p = 0.0009	—	—	—	—	—	—	—	—
LYVE1	↑ p = 0.003	↑ p = 0.0003	No change	↓ p = 0.0004	↑ p = 0.002	↑ p < 0.0001	No change	↓ p < 0.0001	—	—	—	—	—	—	—	—
ANG-2	↑ p = 0.004	↑ p = 0.0002	No change	↓ p = 0.0004	↑ p = 0.0006	↑ p < 0.0001	No change	↓ p < 0.0001	—	—	—	—	—	—	—	—
Brain regions	Dentate gyrus								3rd Ventricle
Cell marker categories
Neurodegeneration
TAU	↑ p < 0.0001	↑ p = 0.016	↓ p < 0.0001	No change	↑ p < 0.0001	↑ p < 0.0001	↓ p < 0.0001	↓ p < 0.0001	—	—	—	—	—	—	—	—
TDP43	↑ p < 0.0001	↑ p = 0.002	↑ p = 0.006	↓ p = 0.002	↑ p = 0.009	↑ p = 0.0008	↓ p = 0.0007	↓ p = 0.0003	—	—	—	—	—	—	—	—
Neuroregeneration
DCX	↑ p = 0.0005	↑ p = 0.006	No change	↓ p = 0.04	↑ p = 0.0005	↑ p < 0.0001	No change	↓ p < 0.0001	No change	No change	↑ p < 0.0001	No change	No change	No change	↑ p = 0.01	No change
Ki67	↑ p = 0.01	No change	↑ p = 0.008	↑ p = 0.007	↑ p = 0.01	No change	↑ p = 0.01	No change	No change	No change	↑ p < 0.0001	No change	↑ p = 0.002	p < 0.0001	No change	↑ p < 0.0001
SOX2	↑ p = 0.004	↑ p = 0.0003	↓ p = 0.04	No change	No change	No change	↑ p = 0.01	↑ p = 0.0002	↓ p < 0.0001	No change	↑ p = 0.009	↑ p = 0.04	↓ p = 0.03	p < 0.0001	No change	No change

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^{^*}

p value Naive versus CCI.

^{^**}

p value CCI versus CCI + GLI.

CCI, controlled cortical impact; TBI, traumatic brain injury; GLI, glibenclamide.

Table 6.

Effect of GLI Treatment in Males and Females on CBF at 21-Day Post-TBI

Male	Cerebral blood flow (PU) ipsilateral (21d) Mean (±SE)	ANOVA or Tukey’s p	Female	Cerebral blood flow (PU) ipsilateral (21 days) Mean (±SE)	ANOVA or Tukey’s p
Naive	325.9 (6.53)	Treatment = 0.002**	Naive	325.7 (2.2)	Treatment = 0.003**
TBI	195.2 (24.17)	Treatment = 0.002**	TBI	318.2 (15.7)	Treatment = 0.003**
TBI + Vehi	193.6 (46.32)		TBI + Vehi	268 (36.4)
TBI + GLI	280.3 (4.3)		TBI + GLI	307.4 (16.2)

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CBF, cerebral blood flow; GLI, glibenclamide; TBI, traumatic brain injury.

**p < 0.01.

In the ipsilateral DG, (Fig. 3Ci) male TDP43 expression was increased 72 h post-CCI (untreated-CCI vs. naive: M.D. = −1.2E6; 95% CI = −1.5E6 to −9.3E5; p < 0.0001). This was further increased with GLI (CCI + GLI vs. untreated-CCI: M.D. = −4.3E5; 95% CI = −7.3E5 to −1.4E5; p = 0.006). In contrast, in females at 72 h post-CCI, TDP43 expression was downregulated with GLI (CCI + GLI vs. untreated-CCI: M.D. = 6.2E5; 95% CI = 3.2E5–9.2E5; p = 0.0007; Fig. 3Cii). Similar TDP43 expression patterns were observed in both males and females at 21-day post-CCI and with GLI treatment. In both sexes, the post-traumatic increase in TDP43 expression at 21-day, was downregulated by GLI (CCI + GLI vs. untreated-CCI: Males: M.D. = 2.2E6; 95% CI = 9.8E5–3.5E6; p_males = 0.002; Females: M.D. = 1.6E6; 95% CI = 9.5E5–2.3E6; p_females = 0.0003) (Fig. 3Ci, ii, Table 5, Supplementary Fig. S3Ai, ii).

We next evaluated TDP43 expression in the ipsilateral thalamus (Fig. 3Di, ii, Supplementary Fig. S4Ai, ii). In males, at 72 h post-CCI, TDP43 expression was increased (untreated-CCI vs. naive: M.D. = −6.5E5; 95% CI = −7.0E5 to −5.9E5; p < 0.0001). GLI treatment reversed increased expression of TDP43 toward normal (CCI + GLI vs. CCI: M.D. = 6.3E5; 95% CI = 5.8E5–6.8E5; p < 0.0001). Conversely, in females at 72 h post-CCI, there was no change in TDP43 expression in the ipsilateral thalamus. At 21-day post-CCI, male thalamic TDP43 expression remained elevated (untreated-CCI vs. naive: M.D. = −8.0E5; 95% CI = −1.0E6 to −5.5E5; p < 0.0001). It continued to be responsive to attenuation with GLI (CCI + GLI vs. CCI: M.D. = −8.0E5; 95% CI = 5.5E5–1.0E6; p < 0.0001). At this 21-day timepoint, unlike at 72 h, female patterns mirrored males, as increased TDP43 expression was observed post-CCI (CCI vs. naive: M.D. = −6.8E5; 95% CI = −9.3E5 to −4.3E5; p < 0.0001). Increased TDP43 expression was downregulated with GLI (CCI + GLI vs. untreated-CCI: M.D. = 9.3E5; 95% CI = 6.8E5–1.1E6; p < 0.0001) (Table 5).

There appeared to be an inverse relationship between 72 h TDP43 expression levels and distance from the CCI-nidus with lowest levels noted in the female thalamus. In most brain regions, TDP43 expression increased further by 21 days. There were two exceptions to this pattern: the male ipsilateral cortex and thalamus. TDP43 levels markedly decreased at 21 days versus 72 h in male ipsilateral cortex after untreated-CCI (Fig. 3B i, ii); this pattern was less pronounced in females. Unlike other brain regions, thalamic TDP43 in females was not increased acutely/at 72 h after CCI but was even higher than male TDP43 by 21 days; the implications of thalamic TDP43 are discussed later instead of therapies such as thalamic deep brain stimulation (DBS) after TBI.⁶⁸

Persistently elevated TAU at 21-day post-CCI noted in females only is decreased by GLI and DMSO, while GLI reduces TAU at 72 h post-CCI across sex

Unlike TDP43, there were no baseline differences in naive TAU levels based on sex or brain region, with minimal expression (Fig. 4Ai, ii, Bi, ii, Ci, ii, Di, ii, Supplementary Figs. S2, S3, and S4).

In males, at 72 h post-CCI, TAU in the ipsilateral cortex was increased (untreated-CCI vs. naive: M.D. = −8.6E6; 95% CI = −1.0E7 to −6.3E6; p < 0.0001). GLI decreased TAU expression (CCI + GLI vs. untreated-CCI: M.D. = 3.7E6; 95% CI = 1.4E6–6.0E6; p = 0.0032) (Fig. 4Bi, Table 5). 21-day post-CCI, TAU expression was downregulated (untreated-CCI vs. naive: M.D. = −1.6E6; 95% CI = −2.0E6 to −1.3E6; p < 0.0001). GLI further reduced TAU expression toward normal (CCI + GLI vs. untreated-CCI: M.D. = 1.2E6; 95% CI = 9.2E5–1.6E6; p < 0.0001). In females, at 72 h post-CCI, a similar expression pattern of TAU was observed (Fig. 4Bii). In contrast to males, at 21-day post-CCI, TAU expression in females remained elevated (untreated-CCI vs. naive: M.D. = −7.3E6; 95% CI = −1.0E7 to −4.2E6; p = 0.0003). It was normalized with GLI (CCI + GLI vs. untreated-CCI: M.D. = 6.6E6; 95% CI = 3.5E6–9.6E6; p = 0.0006).

TAU expression in the DG is shown in Figure 4Ci, ii and Supplementary Figure S3. In males, TAU expression increased at 72 h post-CCI (untreated-CCI vs. naive: M.D. = −2.3E6; 95% CI = −2.8E6 to −1.9E6; p < 0.0001). It was reduced by GLI (CCI + GLI vs. untreated-CCI: M.D. = 1.3E6; 95% CI = 9.1E5–1.8E6; p < 0.0001; Fig. 4Ci, Table 5). A similar pattern was noted in females at: 72 h post-CCI, TAU expression increased (untreated-CCI vs. naive: M.D. = −5.6E6; 95% CI = −6.8E6 to −4.5E6; p < 0.0001). GLI treatment decreased TAU expression (CCI + GLI vs. untreated-CCI: M.D. = 4.0E6; 95% CI = 2.8E6–5.2E6; p < 0.0001; Fig. 4Cii, Table 5). At 21-day post-CCI, TAU expression was decreased in males, whereas in females it remained upregulated; GLI treatment downregulated TAU expression in both sexes.

In the ipsilateral thalamus at 72 h post-CCI, there was no increase in TAU expression in males (Fig. 4Di, Supplementary Fig. S4). However, by 21-day post-CCI, TAU was markedly upregulated (untreated-CCI vs. naive: M.D. = −3.3E6; 95% CI = −4.1E6 to −2.6E6; p < 0.0001). This was reduced by GLI (CCI + GLI vs. untreated-CCI: M.D. = 3.3E6; 95% CI = 2.6E6–4.1E6; p < 0.0001; Table 5). In contrast, in females at both 72 h and 21-day post-CCI TAU was markedly increased (untreated-CCI vs. naive: M.D. = −1.2E6; 95% CI = −1.5E6 to −1.0E6; p < 0.0001; Fig. 4Dii, Supplementary Fig. S4). The expression of TAU at both time points was responsive to GLI with downregulation toward normal (untreated-CCI vs. CCI + GLI: M.D. = 1.1E6; 95% CI = 9.3E5–1.4E6; p < 0.0001; Table 5).

TAU expression at 72 h post-CCI was greatest at the epicenter of injury (ipsilateral cortex), and levels were similarly elevated in males and females versus naive (all p < 0.0001). However, in other brain regions, although TAU was diffusely elevated after untreated-CCI at 72 h, the extent/levels were markedly higher in females. In both sexes and regions (particularly cortex), there appeared to be a stepwise benefit—that is, some benefit from DMSO-vehicle with additional benefit of GLI. As noted above, only females had increased TAU at 21 days in the ipsilateral cortex and DG-GLI (and DMSO-vehicle) normalized this to almost naive levels. Like TDP43, in the thalamus, GLI but not DMSO-vehicle reduced TAU to near baseline naive levels by 21 days across sex (all p < 0.0001).

Sex- and region-specific differences in SOX2 and Ki67 expression post-CCI with or without GLI treatment

Given the potential impact of neurogenesis on recovery/functional outcome after CCI,^69,70 we evaluated neurogenesis markers Ki67, SOX2, and DCX in all groups and both sexes at 72 h and 21-day post-CCI across several brain regions—DG (SGZ; Fig. 5A, C, Supplementary Fig. S5, Table 5) and third ventricle (subventricular zone, site of neurogenesis, Supplementary Figs. S6, Figs. S7).

FIG. 5. — Effect of GLI treatment on expression of Ki67 and SOX2 in ipsilateral DG in males and females at 72 h and 21-day post-CCI Immunofluorescence image of the ipsilateral DG of CCI, CCI + GLI treated males and females mice at 72 h post-CCI, using Ki67 (purple) antibody (Ai, ii). Graphs showing quantification of expression intensity of Ki67 in (DG, Bi, ii) in males and females. Immunofluorescence image of the ipsilateral DG of CCI, CCI + GLI treated male and female mice at 72 h post-CCI, using SOX2 (red) antibody **(C)**. Graphs showing quantification of expression intensity of SOX2 in (DG, Ci, ii) in males and females. All the immunofluorescent sections were counterstained with DAPI nuclear stain (blue). Scale bars 100 μm. Protein expression data were compared using one-way ANOVA followed by Tukey’s post hoc test, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 Naive versus CCI; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 CCI versus CCI + GLI at 72 h and 21-day post-CCI. CCI, controlled cortical impact; GLI, glibenclamide.

In males at 72 h post-CCI, Ki67 expression in the DG was increased (untreated-CCI vs. naive: M.D. = −1.2E5; 95% CI = −2.2E5 to −2.6E5; p < 0.01). It was further upregulated with GLI (CCI + GLI vs. untreated-CCI: M.D. = −1.4E5; 95% CI = −2.4E5 to −4.1E4; p = 0.008; Fig. 5Ai, Bi, Table 5). Similar pattern was observed in females at 72 h post-CCI with increased Ki67 post-CCI, which was further augmented with GLI (CCI + GLI vs. CCI: M.D. = −2.6E5; 95% CI = −4.7E5 to −5.1E4; p < 0.01; Fig. 5Aii, Bii). In both sexes, no change in Ki67 expression was observed in either TBI group with/without GLI treatment at 21 days.

SOX2 expression was increased in the male DG 72 h post-CCI (untreated-CCI vs. naive: M.D. = −8.1E4; 95% CI = −1.3E5 to 3.0E4; p = 0.004). Surprisingly, with GLI treatment it was decreased (CCI + GLI vs. untreated-CCI: M.D. = 5.0E4; 95% CI = 12.34–1.0E5; p = 0.04; Fig. 5C, Di, Table 5). By 21 days, male SOX2 was further upregulated (untreated-CCI vs. naive: M.D. = −2.7E5; 95% CI = −3.9E5 to −1.5E5; p = 0.0003). GLI treatment had no effect on the expression of SOX2. Conversely, in females, at 72 h post-CCI, SOX2 expression did not change compared with naive (Fig. 5C, Dii, Table 5), but was increased by GLI (CCI + GLI vs. untreated-CCI: M.D. = −1.5E5; 95% CI = −2.7E5 to −3.2E4; p < 0.01). Similarly, female SOX2 expression did not change even at 21-day post-CCI, but once again, this regenerative marker was increased with GLI (CCI + GLI vs. untreated-CCI: M.D. = −3.9E5; 95% CI = −5.4E5 to −2.3E5; p = 0.0002). No differences were noted in the expression of either marker regardless of time, sex, or treatment in the third ventricle (Supplementary Figs. S6, Figs. S7).

Expression of DCX showed a similar pattern in ipsilateral DG, post-CCI, and with GLI in both males and females at acute (72 h) and chronic (21 days) time points (Supplementary Fig. S7, Ai, ii, Bi, ii). At 72 h post-CCI, expression of DCX showed a significant increase in both males and females, while GLI treatment produced a decreasing trend. At 21-day post-CCI, expression of DCX was decreased in both males and females, and GLI treatment further downregulated its expression across sexes.

Similar benefit of GLI in restoring CBF across sex at 21-day post-CCI, while DMSO-vehicle showed pronounced benefit in females

As expected,^71,72 CBF was decreased post-CCI, in the ipsilateral hemisphere at 24 h and 72 h in both male and female mice (Fig. 6A, B; Table 6). In males, although there was no acute recovery of CBF with GLI treatment, by 21 days, it dramatically improved versus untreated mice (p = 0.002); GLI treatment outperformed vehicle, although it did not return values to baseline (Fig. 6A). In contrast, in female mice, although GLI improved CBF by 21 days versus untreated-CCI (p = 0.002), the benefit was similar to DMSO-vehicle treatment (Fig. 6B).

To interrogate molecular markers potentially associated with increased CBF after GLI treatment, we evaluated the expression of VEGFA, ANG-2, and LYVE1 (Figs. 2i, ii, 7,8). In both sexes, VEGFA expression at 72 h was increased post-CCI, which was further increased with GLI (CCI + GLI vs. untreated-CCI: Male: M.D. = −2.3E6; 95% CI = −3.0E6 to −1.7E6; p_male < 0.0001; Female: CCI + GLI vs. untreated-CCI: M.D. = −1.9E6; 95% CI = −3.0E6 to −7.5E5; p_female < 0.01, Fig. 7Bi, ii). VEGFA expression continued to increase across sexes post-CCI, even at 21 days (all p < 0.0001), with higher levels noted in males. However, at 21 days, both GLI and DMSO-vehicle treatment were associated with lower levels versus untreated-CCI (all p < 0.0001). The improved CBF at 21 days with GLI treatment in males is thus associated with an earlier but nonpersistent increase in VEGFA.

LYVE1 and ANG-2 showed similar expression patterns. Both LYVE1 and ANG-2 were only detectable in the ipsilateral cortex post-CCI where the expression was increased versus naive at 72 h in both males (CCI vs. naive: LYVE1: M.D. = −1.4E6; 95% CI = −2.3E6 to −5.7E5; p_{male_LYVE1} = 0.003; CCI vs. naive: ANG-2: M.D. = −1.4E6; 95% CI = −2.3E6 to −5.40E5; p_{male_ANG-2} <0.01) and females (CCI vs. naive: LYVE1: M.D. = −2.1E6; 95% CI = −3.3 to −8.8E5; p_{female_LYVE1}=0.002, CCI vs. naive: ANG-2: M.D. = −2.3E6; 95% CI = −3.3E6 to −1.20E6; p_{female_ANG-2}<0.001) (Fig. 8Bi, ii, Di,ii). GLI did not alter the expression of either marker at 72 h, however, in females treated with DMSO-vehicle both LYVE1 and ANG-2 were markedly reduced (CCI + vehi vs. CCI: LYVE1: M.D. = 1.6E6; 95% CI = 4.4E5–2.9E6; p_{female_LYVE1}=0.01, CCI + vehi vs. CCI: ANG-2: M.D. = 1.7E6; 95% CI = 7.0E5–2.8E6; p_{female_ANG-2}=0.003). By 21 days in both sexes, levels of both markers had markedly decreased without treatment and were further reduced to naive levels by GLI (Fig. 8).

FIG. 8. — Effect of GLI treatment on expression of ANG-2 and LYVE1 in ipsilateral cortex in males and females at 72 h and 21-day post-CCI Immunofluorescence image of ipsilateral cortex of CCI, CCI + GLI treated males and females mice at 72 h post-CCI, using LYVE1 (purple) **(A)** and ANG-2 (green) and antibodies **(C)**. Graphs showing quantification of expression intensity of LYVE1 (Bi, ii) and ANG-2 (Di, ii) in males and females. All the immunofluorescent sections were counterstained with DAPI nuclear stain (blue). Scale bars 100 μm. Protein expression data were compared using one-way ANOVA followed by Tukey’s post hoc test, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 Naive versus CCI; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 CCI versus CCI + GLI at 72 h and 21-day post-CCI. CCI, controlled cortical impact; GLI, glibenclamide.

FIG. 7. — Effect of GLI treatment on expression of VEGFA in ipsilateral cortex in males and females at 72 h and 21-day post-CCI Immunofluorescence image of ipsilateral cortex of CCI, CCI + GLI treated males and females mice at 72 h post-CCI, using VEGFA (green) antibody (Ai, ii). Graphs showing quantification of expression intensity of VEGFA (Bi, ii) in males and females. All the immunofluorescent sections were counterstained with DAPI nuclear stain (blue). Scale bars 100 μm. Protein expression data were compared using one-way ANOVA followed by Tukey’s post hoc test, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 Naive Vs CCI; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 CCI versus CCI + GLI at 72 h and 21-day post-CCI. CCI, controlled cortical impact; GLI, glibenclamide.

Discussion

There are currently no approved therapies that improve functional outcomes after TBI. In part, this is attributed to significant TBI heterogeneity and limitations of a one-size-fits-all approach in the existing clinical guidelines. An important contributor to TBI heterogeneity is biological sex. Here, we present results from the first murine randomized and blinded testing of the promising drug GLI that quantifies its effects on key sexually dimorphic behavioral, molecular, and physiological responses in a pre-clinical model of TBI.

We report four main findings: (1) There was a range of sexual dimorphism in several functional, molecular, and physiological measures at baseline, post-CCI, and in response to GLI treatment. (2) In both sexes, GLI improved multiple metrics of functional outcome and CBF; however, benefits were nuanced and sex-specific. There was greater cognitive, motor, and CBF benefit in males. (3) There was variable expression of molecular markers (neurodegeneration, neurogenesis, angiogenesis) post-CCI with and without GLI by sex, brain region, and time. Although GLI reduced markers of neurodegeneration and increased markers of neurogenesis and angiogenesis in both sexes, temporal, spatial, and marker-specific differences were noted in males versus females. For example, TAU levels remained markedly increased in females only at 21 days and were reduced with GLI. 4) DMSO-vehicle treatment had previously unrecognized effects in females—it afforded benefit post-CCI (occasionally equivalent to GLI) in some measures of cognitive function, CBF, and reduced neurodegenerative marker expression.

Sex, GLI, and functional outcome

Our data confirmed prior pre-clinical reports showing that TBI resulted in similar cognitive impairment across sex.^62,73,74 Although GLI improved cognitive function in both sexes, the benefit was more pronounced in males. This is directionally consistent with prior work demonstrating that genetic loss of Abcc8 (encoding SUR1) strongly protected against post-traumatic brain atrophy in male, but not female mice.⁸ Our data did not identify a motor deficit in untreated males post-CCI, unlike prior work.^62,75 This may be due to differences in injury model, severity, or outcome measure. Nonetheless, treatment with GLI post-CCI augmented motor performance in both sexes, again with a greater benefit noted in males; GLI-treated males outperformed naive mice. The finding that GLI-treated males post-CCI outperformed naive on the rotarod task is intriguing and remains unexplained. It is possible that since no rotarod motor deficit was noted post-CCI, the increased motor performance after GLI is related to an impact on psychomotor behaviors like anxiety (related to loss of inhibition) rather than purely motor function. This would need further investigation in future studies. The results of our study align with existing literature that has observed similar delayed motor coordination deficits in the pole-test performed until 9 months post-TBI.⁷⁶ In this study, no motor deficit was observed in male mice till 4.5 months post-TBI as compared to naive and sham groups.

Regarding psychological outcomes, TBI induced more pronounced anxiety in males versus females, as shown by a reduced amount of time in the anxiogenic regions of the EPM.^57,77,78 Recent studies in male rats support our findings that TBI exhibits substantially increased anxiety, impulsivity, and in attention, highlighting the injury’s profound impact on behavioral regulation.^79,80 The anxiolytic effect of GLI was equivalent in both males and females.

Sex, GLI, and markers of neurodegeneration

TBI is an important risk factor for accelerated cognitive decline, including neuropathological hallmarks of AD, or TAU and TDP43 proteinopathies.^{43,45–47,49,52,81–84} Sex-based differences post-TBI in cognitive decline remain poorly understood. There are few prior reports suggesting that increased deficits occur in male mice after rotational and repeat concussive injury, while others suggest equivalence across sex.^62,73,75 Here we report sexually dimorphic and spatio-temporally variable upregulation of TDP43 and TAU in the cortex, DG, and thalamus evident by 72 h post-CCI. Both proteins colocalized spatially at 72 h and 21 days post-CCI, as seen in other neurodegenerative disease^85–88 suggesting mechanistic synergy. Although TDP43 and TAU were upregulated across sex and may contribute to the observed behavioral deficits, expression was greater in males at 72 h but in females increased expression was observed at 21 days. TAU expression was much more pronounced in females versus males in several brain regions and persisted up to 21 days, identifying a potentially sex-specific therapeutic target. To the best of our knowledge, there is a lack of prior research on the expression of TDP43 and TAU post-CCI in males and females at 72 h and 21 days. Notably, previous studies have shown that administration of testosterone post-CCI can mitigate neuronal degeneration by modulating mitochondrial dysfunction⁸⁹ and promote neuronal survival.⁹⁰ These studies may provide a plausible explanation for the observed decrease in expression of TDP43 and TAU in males. Our findings align with recent studies demonstrating that female mice exhibit robust protective response to TBI,⁹¹ characterized by reduced acute cell loss, whereas male mice show exacerbated astrogliosis, neuronal death, and enlarged lesion volume up to 7 days post-TBI.⁷ Females in our study showed a delayed onset of neurodegeneration, which may be due to neuroprotective effects of estrogen.⁹² This notion is supported by numerous studies demonstrating that estrogens exert beneficial effects on neurodegenerative diseases, specifically Alzheimer’s and Parkinson’s.^93,94 Additionally, estrogen has been shown to reduce disease progression, alleviate symptoms, enhance cognitive function, and protect against neuronal damage.

TDP43 was increased in the thalamus in females at 21 days. Emerging data suggest a pivotal role of the thalamus in post-CCI pathobiology, which represents a therapeutic target. In some cases, DBS has reversed chronic cognitive deficits in TBI survivors.^68,95,96 To our knowledge, there are no prior reports identifying sexual dimorphism. Understanding the role of thalamic TDP43 in chronic post-traumatic neurodegeneration may help identify future targeted therapies, especially in females.

Our data may provide new insight into sex-specific neurodegenerative pathways post-TBI that warrant further investigation, given the role of these proteinopathies in cognitive dysfunction. Interestingly, the benefit of GLI was not sexually dimorphic, it reduced the expression of TDP43 and TAU in both sexes. However, DMSO-vehicle treatment had a surprisingly similar benefit to GLI in females post-CCI (discussed below).

Sex, GLI, and markers of neurogenesis

Increased neurogenesis has been reported post-TBI,^69,97–100 and thought to be important for replacing dying/degenerating neurons and stabilizing the neuronal network.¹⁰¹ GLI promotes neurogenesis in other diseases like ischemic stroke.^27,29,102 However, these studies have been in males with little exploration of sex-based differences in neurogenesis or response to treatment. We used SOX2 (neural progenitor cell marker), Ki67 (proliferation marker), and DCX (immature granule cell marker, evaluated in the DG subgranular zone) to explore neurogenesis post-CCI and response to GLI in several brain regions across sexes.

There was marked sexual dimorphism in the expression of neurogenesis markers and response to GLI: SOX2 expression was higher in the male DG (with no further increase after GLI treatment at 21 days), whereas expression after untreated-CCI was low in females and increased (to male levels) with GLI by 21 days. We speculate that this lack of endogenous neurogenesis suggests a reduced compensatory response to TBI in females, which can be augmented by GLI, thus identifying a potentially unique therapeutic opportunity in females to improve DG (and cognitive) recovery. Expression of Ki67 and DCX was similar across sexes: the increase in these markers was transient with or without GLI treatment-noted only at 72 h. The acute benefit of GLI in increasing neural progenitor cells might underlie some of its cognitive benefit, supporting the hypothesis that neurogenesis in DG is maintained by precursor cells with finite proliferative capacity.^70,103

Sex, GLI, and CBF

TBI can result in cerebrovascular injury that may manifest CBF changes, blood–brain barrier damage, and microhemorrhage^104–106 resulting in TAU accumulation¹⁰⁷ and chronic neurodegeneration.¹⁰⁸ A cerebrovascular link has been suggested between TBI and neurodegenerative disease.⁴⁶ An acute reduction in CBF with delayed recovery has been reported,^109–112 with increasing clinical interest in CBF augmentation; however, much of these therapies focus on acute TBI in the context of disrupted autoregulation, ischemia, and intracranial hypertension. Few studies have evaluated the impact of therapies on delayed CBF, although flow reductions linked to tissue loss have been reported at 1 year after CCI in rats.¹¹³ Here, GLI did not impact CBF acutely, although untreated CBF improved with time, equivalent benefit with GLI treatment was noted by 21 days in both males and females.

Molecularly, this improvement in CBF was preceded by VEGFA (angiogenesis), ANG-2 (angiogenesis), and LYVE1 (lymphangiogenesis) expression. VEGFA has been noted to increase within 12 h post-TBI^114,115 and promotes neurogenesis.^115–118 We confirmed that even in untreated mice at 72 h after CCI, all three markers were increased versus naive, with no significant sex-based differences. This was further augmented by GLI across sex. Surprisingly, VEGFA expression at 21 days remained increased after untreated-CCI (male > female), but was downregulated with GLI (less in males). It is possible that the greater benefit of GLI in restoring male CBF is related to early VEGF-pathways, followed by obviating the need for delayed angiogenesis. A similar pattern was true for ANG-2 and LYVE1; however, these markers were primarily noted in the peri-contusion, and unlike VEGFA, both were downregulated by 21 days after untreated-CCI. Future studies should evaluate sex-based differences in the impact of these pathways on the timing of angiogenesis, neuroinflammation, and necrotic tissue clearance; this may further inform secondary injury processes like cerebral edema and/or restoration of physiological homeostasis.

Reductions in CBF have also been associated with neuropathological accumulation of TAU,^119,120 further supporting a cerebrovascular link between TBI and accelerated cognitive decline. Our data support this; however, in our study, it remains unclear whether these are causally linked: acutely, increased TAU is associated with decreased CBF across sex. By 21 days after CCI, TAU decreases and CBF increases even in untreated mice—this is further improved by GLI, suggesting another potential mechanism by which GLI may benefit post-CCI cognitive decline—preferentially in males. This speculation warrants further evaluation. The benefit of DMSO-vehicle on female CBF post-CCI was similar to GLI and unexpected (see below).

Sex, DMSO, and TBI

In several functional, molecular, and physiological outcome measures, particularly in females, CCI + DMSO-vehicle also showed benefit. DMSO is the standard vehicle for pre-clinical studies of GLI.^{9,11,13,19,21,23} Historically, in TBI patients, DMSO has been used to treat intracranial hypertension and cerebral edema.^121–124 In one pre-clinical report, DMSO was proposed as a neuroprotectant with reduced secondary cell death.¹²⁵ Although speculative, it has been proposed that DMSO may prevent mitochondrial damage via an antioxidant capacity to scavenge reactive oxygen species and prevent lipid peroxidation.^121–123 There have been no previous reports on sexual dimorphism in response to DMSO in TBI with greater benefit in females, given the consistency of this surprising finding in our data, it warrants further research.

Strengths and limitations

A strength of our study is the randomized blinded analysis, large sample size, and analysis of sex-based differences post-CCI and GLI treatment at different time points post-TBI, which is novel. Our study includes several behavioral tests, which were subsequently interrogated by physiological and molecular metrics. Our CCI model mimics human contusional TBI and is relevant for patients being enrolled in clinical trials. We now link, for the first time, sex-based differences in TBI and response to GLI to a spectrum of behavioral, molecular, and physiological measures. However, a limitation is that detailed molecular evaluations (e.g., of additional markers like p-TAU, beta-amyloid) remain to be explored. These would be valuable for future studies exploring sex-specific neurodegenerative pathways. Additionally, in our study, GLI was administered 10 min post-CCI. This timeline was selected to maintain consistency with prior pre-clinical evaluations of GLI in TBI.^9,11,13 Clinical trials of GLI in TBI also require rapid administration of the drug within 6.5 h post-TBI,¹ the conversion of this timeline to murine injury is unclear. While there is no universal conversion, the rodent time scale does appear abbreviated in TBI,¹²⁶ suggesting that 10-min post-TBI is a longer timeframe in humans. Nonetheless, this important limitation should be addressed in future research to investigate benefits from GLI treatment at clinically relevant time points to better understand its therapeutic potential. To mimic the clinical spectrum of TBI that occurs in females across all stages of the estrus cycle, female mice were not injured at a specific cycle phase. Omission of estrus phase recording and assessment in female mice in this study is an important limitation, since the estrus cycle can influence various behavioral and physiological outcomes.

We used LSCI to assess CBF. While it is a powerful, noninvasive, full-field optical imaging tool, it measures relative blood flow velocity using the changes in the reflection patterns depending on the speed of the erythrocytes in the vessel.¹²⁷ LSCI is also limited to the assessment of superficial flow velocity, and our findings should be confirmed with methods that quantify CBF.

Conclusion

To the best of our knowledge, this is the first study providing a comprehensive overview of multiple interrelated processes that are altered post-TBI and the effects of GLI across sex. We report novel baseline sex-based differences post-TBI and response to GLI measured by behavioral, molecular, and physiological metrics. The multifaceted benefit of GLI is more pronounced in males (particularly in cognitive function). We also identify previously unrecognized benefits of DMSO in females on multiple metrics (MWM, TDP43, and TAU reduction, CBF). Delayed TAU-related pathobiology and thalamic TDP43 may be more prominent in females—both are responsive to GLI. Collectively, our findings may have important implications for future mechanistic evaluations of sex-based differences in GLI-responsive neurodegeneration, neurogenesis, physiology, and functional outcome, as well as study design and analysis plans for clinical trials evaluating GLI in TBI.

Transparency, Rigor, and Reproducibility

In the proposed study, to evaluate the expression of memory formation related proteins post-TBI and with GLI treatment in male versus female sample size was calculated by power analysis using G*power. Based on the conservative estimate of a 30% (Standard Deviation (SD) 15%) difference in male versus female expression, a total sample size of 124 mice was needed for alpha = 0.05, power = 80%. In our study, we have used (120 mice total) n = 6/group/timepoint for behavior and n = 3/group/timepoint for CBF studies. Mice were block randomized into four groups. All experiments and procedures were approved by the Institutional Animal Care and Use Committee at the Barrow Neurological Institute (Phoenix)/St. Joseph’s Hospital and Medical Center (Phoenix), and mice were housed in the Barrow Neurological Institute vivarium, accredited by the AAALAC. Mice were acclimated to the housing facility for at least 1 week before use. Experiments were randomized, and outcomes were obtained by investigators blinded to both treatment group and sex. No mortality was observed after CCI surgery or treatment. None of the mice were excluded in behavioral, CBF, and immunofluorescence studies. All antibodies used in the present study were purchased from commercial sources and have been previously validated for specificity in immunofluorescence applications by the manufacturer. We have not undertaken specific antibody validation experiments in the present study.

The authors agree to provide the full content of the article upon request by contacting the corresponding author.

Supplementary Material

Supplementary Data

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Supplementary Figure S1

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Supplementary Figure S2

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Supplementary Figure S3

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Supplementary Figure S4

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Supplementary Figure S5

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Supplementary Figure S6

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Supplementary Figure S7

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Supplementary Figure S8

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Supplementary Figure S9

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Supplementary Table S1

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Supplementary Table S2

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Supplementary Table S3

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Data Availability

Data supporting the findings of this study are available on appropriate request from the study authors.

Authors’ Contributions

A.R.: Conceptualization, methodology, validation, data collection, investigation, funding acquisition, formal analysis, writing—original draft, visualization. S.P.R.: Methodology, validation, formal analysis, review, and editing. W.Y.: Data analysis, data curation, review, and editing. Saif A., A.E., M.M., J.H., J.C., B.E.Z., S.D., R.A.M.J., Semeon A., E.A.M., G.K., D.R., M.F.W., and A.D.: Review and editing. V.A.V. and K.L.J.F.: Methodology, review, and editing. S.W.C.: Methodology and writing—review and editing. A.J., A.K., and C.L.P.: Writing—review and editing. P.M.K.: Conceptualization, writing—review and editing, supervision, funding acquisition. R.M.J.: Conceptualization, supervision, writing—review and editing, project administration, funding acquisition, resources, and validation.

Author Disclosure Statement

The authors declare no competing interests.

Funding Information

The authors are grateful to funding from the National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS: 1R01NS115815-03, R21NS131689-01 to R.M.J.), Barrow Neurological Foundation (OSRA: 24-500-459-30-09; R.M.J.), Chuck Noll Foundation (OSRA: 23-500-184-70-09; R.M.J. and P.M.K.), and the Barrow Neurological Institute Translational Neuroscience Trainee Fast Track grant (OSRA: 17-0299-30-09; A.R.).

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Supplementary Materials

Supplementary Data

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Supplementary Figure S1

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Supplementary Figure S2

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Supplementary Figure S3

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Supplementary Figure S4

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Supplementary Figure S5

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Supplementary Figure S6

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Supplementary Figure S7

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Supplementary Figure S8

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Supplementary Figure S9

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Supplementary Table S1

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Supplementary Table S2

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Supplementary Table S3

neu.2024.0232_supplementary_tables3.xlsx^{(22.3KB, xlsx)}

Data Availability Statement

Data supporting the findings of this study are available on appropriate request from the study authors.

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PERMALINK

Sexually Dimorphic Responses Reveal Multifaceted Benefits of Glibenclamide in Traumatic Brain Injury

Anupama Rani

Sudhanshu P Raikwar

Wonsuk Yoo

Saif Ahmad

Vincent A Vagni

Keri L Janesko-Feldman

Shaun W Carlson

Adam Eberle

Margaux Miller

John Helm

Joshua Catapano

Benjamin E Zusman

Shashvat Desai

Raemier Anne M Javelosa

Semeon Afework

Erin Audrey McNally

Gary Kohanbash

Dhivyaa Rajasundaram

Michael F Waters

Andrew Ducruet

Ashutosh Jadhav

Aditya Kumar

Chia-Ling Phuah

Patrick M Kochanek

Ruchira M Jha

Abstract

Introduction

Materials and Methods

Animals

Experimental groups

Controlled cortical impact

Drug/vehicle administration

Morris water maze testing

Rotarod testing

Elevated plus maze testing

Cerebral blood flow assessment

Brain processing for immunofluorescence assessments

Immunofluorescence

Statistical analysis

Results

Sexually dimorphic functional deficits post-CCI improved by GLI treatment

GLI-treated males had greater benefit than females on MWM post-CCI

FIG. 1.

Table 1.

Table 2.

GLI improves rotarod deficits post-CCI in females and augments male performance beyond naive levels in males

Table 3.

GLI reduced anxiety measured by EPM across sex post-CCI

Table 4.

Sex- and region-specific differences in TDP43 and TAU expression post-CCI with or without GLI treatment

FIG. 2.

Decreased 21 days DG, cortical, and thalamic TDP43 in GLI-treated mice across sex

FIG. 3.

Table 5.

Table 6.

Persistently elevated TAU at 21-day post-CCI noted in females only is decreased by GLI and DMSO, while GLI reduces TAU at 72 h post-CCI across sex

FIG. 4.

Sex- and region-specific differences in SOX2 and Ki67 expression post-CCI with or without GLI treatment

FIG. 5.

Similar benefit of GLI in restoring CBF across sex at 21-day post-CCI, while DMSO-vehicle showed pronounced benefit in females

FIG. 6.

FIG. 8.

FIG. 7.

Discussion

Sex, GLI, and functional outcome

Sex, GLI, and markers of neurodegeneration

Sex, GLI, and markers of neurogenesis

Sex, GLI, and CBF

Sex, DMSO, and TBI

Strengths and limitations

Conclusion

Transparency, Rigor, and Reproducibility

Supplementary Material

Data Availability

Authors’ Contributions

Author Disclosure Statement

Funding Information

References